The observation by Habu et al. in 1987 that linked the ensuing development of T cells to V(D)J recombination set the stage for efforts in the early 1990s to determine how that linkage was enforced (1). During that time, the identification of the pre-BCR and the demonstration that it was essential for normal B cell development gave rise to speculation that early T lineage progenitors might use an analogous pre-receptor complex (2, 3, 4, 5, 6, 7). Genetic evidence supporting the function of such a complex, as well as biochemical studies supporting its expression on the surface of developing T cells, was beginning to accumulate. The search for the key component of a pre-TCR complex culminated with the cloning and characterization of the pre-Tα (pTα) gene by the von Boehmer laboratory (8), the subject of this month’s Pillars of Immunology article.

Among the initial approaches, the use of transgenic mice expressing TCRβ subunits revealed that ectopic expression of TCRβ could terminate TCRβ rearrangement, induce TCRα rearrangement, and promote development of CD4−8− double negative (DN)2 progenitors to the CD4+8+ double positive stage (9, 10). Moreover, the ability of a TCRβ transgene to promote development was retained when expressed on a mutant SCID or Rag-deficient background, effectively ruling out any partner chains that required gene rearrangement (10, 11, 12). Finally, in a landmark paper from the Hayday laboratory, it was demonstrated that the proportion of developing T cells with in-frame TCRβ rearrangements was enriched as cells differentiated from the CD44−CD25+ DN3 stage to the CD44−CD25− DN4 stage (13). This suggested that the production of a functional TCRβ protein was necessary for development from the DN3 to the DN4 stage, a process that later became known as β-selection (14). These observations were bolstered by biochemical identification of a TCRβ-containing complex expressed in developing T cells that was likely to be responsible for these effects (10).

The von Boehmer laboratory was the first to report the presence of a “pre” receptor complex expressed on the surface of T lineage precursors (10). This complex was initially thought to lack all other TCR components including CD3 (10). After some initial confusion, it was quickly found that this complex had the curious feature of containing dimeric TCRβ subunits in association with CD3 but not TCRα (15). This observation was corroborated by a number of other groups and was generally interpreted to mean that this pre-receptor likely contained TCRβ homodimers (11, 15, 16, 17). This viewpoint was held primarily because no other disulfide-linked TCR subunit could be identified by the surface iodination approaches that were in use at that time. However, this interpretation ran counter to two widely accepted sets of observations. First, it was already clear that, by analogy, early B cell progenitors used a pre-BCR that had a surrogate partner chain comprising two subunits, λ5 and Vpre-B (4, 5, 6). Secondly, work from the laboratories of Klausner, Terhorst, and others had clearly established that mature αβTCR complexes could only be transported to the cell surface if all six TCR subunits (α, β, γ, δ, ε, and ζ) were assembled together as a complete complex (18, 19, 20). Partially assembled subunits were shown to be retained intracellularly and degraded in a location determined by their subunit composition (21, 22, 23). This led to two possible explanations: either immature thymocytes played by different rules or there was an additional, as yet unknown, surrogate TCR subunit encoded in the germline.

The first evidence suggesting there was in fact a surrogate partner chain for TCRβ in the pre-receptor complex came as part of an elegant study by the von Boehmer laboratory seeking to provide evidence in support of their model that pre-receptors comprise TCRβ homodimers (16). Cell lines expressing two β-chains distinguishable by their V regions showed no evidence for β-β pairing, prompting further experimentation to search for partner chains by metabolic labeling. These efforts did reveal the intracellular association of TCRβ with a 33-kDa glycoprotein, initially called gp33. However, in order for gp33 to be the TCRβ partner in the pre-receptor complex, evidence had to be provided for gp33-TCRβ association on the cell surface. That was made possible by a surface biotin-labeling technique developed in the Karjalainen laboratory, which finally provided evidence for surface expression in cell lines of a pre-receptor comprising gp33-TCRβ dimers associated with the CD3 complex (16, 24). In the end, one critical reason why gp33 had escaped detection for so long was its failure to be labeled by surface iodination. It was the biochemical identification of gp33 that set the stage for cloning of the pTα gene about 1 year later in 1994 (8).

The description of the cloning of the pTα gene and characterization of its expression pattern in developing thymocytes in the paper by Saint-Ruf et al. represents a Pillars of Immunology article because it was not only the end of a long quest to identify this elusive TCRβ partner, but it also provided explanations for past observations as well as made possible future genetic approaches to tie the function of the TCRβ-containing pre-receptor to the pTα-containing pre-TCR complex in particular (8). Saint-Ruf et al. identified the pTα gene using degenerate oligonucleotides derived from the microsequencing of pTα peptides. The pTα gene had features one would expect for a TCR subunit. It had a single Ig gene superfamily domain representing a constant domain-like structure. Interestingly, unlike the pre-BCR, there was no domain filling in for a V region. The charged residues in the transmembrane domain of pTα were typical of those involved in TCR subunit pairing. Finally, pTα had an extensive cytoplasmic tail with motifs suggesting a role in signaling. Analysis of the expression pattern of pTα mRNA revealed that its expression did not require V(D)J recombination and was restricted to developing thymocytes through the double positive stage, but not beyond. This suggested that pre-receptor complexes containing TCRβ were expressed in developing thymocytes not because immature thymocytes failed to obey the rules of TCR assembly and transport established for mature T cells, but rather occurred because they expressed the additional subunit (pTα) that was needed to complete the receptor. Interestingly, there did appear to be some developmental regulation of pre-TCR expression beyond that responsible for expression of the pTα subunit. Transfection of pTα and TCRβ into a TCR-deficient hybridoma used to model a mature T cell failed to support surface expression (8). This suggested that either pTα-β dimers are actively retained within mature T cells or that they fail to be transported to the cell surface because of the absence of a putative additional pre-TCR subunit, perhaps a VpreT-like component. Despite repeated attempts to identify a VpreT equivalent (corresponding to VpreB), none has been identified.

This foundational article made possible the ensuing 15 years of research focused on understanding the molecular basis for how the pre-TCR controls early thymocyte development. Germline ablation of the pTα gene revealed that it was essential for development of αβ-lineage cells, genetically linking the pTα-containing pre-TCR to the process of β-selection and αβ-lineage differentiation (25). This, in turn, enabled dissection of the pTα domains required for function, including exodomain deletion studies that led to the proposal that the pre-TCR complex does not require a ligand for signaling (26, 27), and to mapping studies that established the basis for the silencing of surface pre-TCR expression upon competitive displacement of pTα by TCRα (28). These approaches culminated in a relatively recent study suggesting that pTα-driven oligomerization is responsible for the ability of the pre-TCR complex to signal without ligand (29). Numerous questions remain including how signaling by a ligand-independent receptor is terminated and whether the discovery of pTα represents a completion of the cataloguing of pre-TCR subunits or whether an additional subunit or subunits remains to be identified.